MUSSEL ADHESIVE PROTEIN-BASED PHOTOTHERMAL AGENT AND PHOTOTHERMAL- RESPONSIVE ADHESIVE NANOPARTICLES

Abstract

The present invention relates to: a photothermal agent which includes a mussel adhesive protein; and photothermal-responsive nanoparticles that generate a biocompatible gas by means of light and heat and release a drug. Nanoparticles according to the present invention exhibit a photothermal effect when near-infrared rays are applied thereto, and may be applied to trimodality therapy in which a biocompatible gas is generated by means of light and heat to induce the release of a drug.

Claims

1. Nanoparticles comprising a mussel adhesive protein, A, and MX.sub.3, wherein A is one selected from the group consisting of S-nitrosogiutathione, N,N′-di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine, Roussin's black salt, and S-nitrosothiol (SNO), M is Fe or V, and X is F, Cl, Br, or I.

2. The nanoparticles of claim 1, wherein A generates a gas in response to light and heat,

3. The nanoparticles of claim 1, wherein the mussel adhesive protein is a protein consisting of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or a fusion protein to which one or more amino acid sequences selected from the group are linked.

4. The nanoparticles of claim 3, wherein the fusion protein is a fusion protein consisting of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 15.

5. The nanoparticles of claim 3, wherein the mussel adhesive protein is characterized in that 10 to 100% of the total tyrosine residues are modified into DOPA.

6. The nanoparticles of claim 5, wherein the nanoparticles include a DOPA-metal complex.

7. The nanoparticles of claim 6, wherein the nanoparticles have a photothermal conversion ability in the near-infrared region.

8. The nanoparticles of claim 1, wherein the nanoparticles have biocompatibility.

9. The nanoparticles of claim 1, further comprising an anti-cancer drug.

10. The nanoparticles of claim 9, characterized in that a gas is generated by a photothermal effect.

11. The nanoparticles of claim 10, wherein the gas is any one or more selected from the group consisting of nitric oxide, oxygen, hydrogen, carbon monoxide, hydrogen sulfide, sulfur dioxide, carbon dioxide, DL-menthol, and perfluorocarbon.

12. A method for treating cancer, comprising a step of: administering the nanoparticles according to claim 1 in a therapeutically effective amount to a subject in need thereof.

13. The method of claim 12, wherein the nanoparticles further comprise an anti-cancer drug, wherein the anti-cancer is any one or more selected from the group consisting of doxorubicin, paclitaxel, azithromycin, erythromycin, vinblastine, bleomycin, dactinomycin, daunorubicin, idarubicin, mitoxantrone, plicamycin, and mitomycin.

14. The method of claim 13, wherein the anti-cancer drug is doxorubicin.

15. The method of claim 13, wherein the anti-cancer drug is supported on the nanoparticles.

16. (canceled)

17. (canceled)

18. A method of preparing nanoparticles comprising: a step 1) of mixing a mussel adhesive protein and MX.sub.3, wherein M is Fe or V, and X is F, Cl, Br, or I; and a step 2) of electrospraying the mixture of the step 1) at a rate of 0.5 to 1.5 ml/h and a voltage of 5 to 15 kV.

19. The method of claim 18, wherein A is mixed together with the mussel adhesive protein and MX.sub.3 in the step 1), wherein the A is one or more selected from the group consisting of S-nitrosogiutathione, N,N′-di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine, Roussin's black salt, and S-nitrosothiol (SNO).

20. The method of claim 19, wherein an anti-cancer drug is mixed together with the mussel adhesive protein, MX.sub.3 and, A in the step 1), wherein the anti-cancer drug is any one or more selected from the group consisting of doxorubicin, paclitaxel, azithromycin, erythromycin, vinblastine, bleomycin, dactinomycin, daunorubicin, idarubicin, mitoxantrone, plicamycin, and mitomycin.

21. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is a schematic diagram showing that photothermal effects are caused through near-infrared rays to induce annihilation of the cancer cells when applying a photothermal agent based on a mussel adhesive protein and nanoparticles containing a photothermal-responsive gas donor and a drug according to the present invention to cancer cells, and complex anti-cancer effects are obtained by inducing the generation of a nitric oxide gas, thereby releasing the drug.

[0057] FIG. 2 is scanning electron microscope (SEM) images of nanoparticles (FeMAP NPs, VMAP NPs) prepared by forming a metal-catechol complex through iron (Fe) ions and vanadium (V) ions, i.e., metal ions.

[0058] FIG. 3, as results of measuring adhesion values of the respective nanoparticles according to Example 1 using QCM, shows results of comparing the measured adhesion values of the nanoparticles by using bovine serum albumin (BSA) nanoparticles as a control group.

[0059] FIG. 4 is a graph showing comparison results obtained by comparing UV absorption spectrums of the respective nanoparticles according to Example 2 using deionized water (DW), FeCl.sub.3, VCl.sub.3, and a mussel adhesive protein (MAP) as a control group.

[0060] FIG. 5 is a graph measuring temperature values generated when applying near-infrared rays to the respective nanoparticles according to Example 2.

[0061] FIG. 6 is an SEM image of nanoparticles (VMAP@GSNO NPs) containing S-nitrosoglutathione (GSNO) that is a nitric oxide gas donor according to Example 3.

[0062] FIG. 7 is a graph measuring the generation of a nitric oxide gas of VMAP@GSNO nanoparticles according to Example 3. GSNO was used as a control group.

[0063] FIG. 8 is an SEM image of nanoparticles (VMAP@GSNO/DOX nanoparticles) containing GSNO and DOX according to Example 4.

[0064] FIG. 9 shows a graph measuring the drug release of VMAP@GSNO/DOX nanoparticles according to Example 4.

[0065] FIG. 10 shows a graph of MCF7 cell viability when near-infrared rays are not applied in Example 5.

[0066] FIG. 11 shows a graph of MCF7 cell viability according to time values at which near-infrared rays are applied in Example 5.

MODES OF THE INVENTION

[0067] Hereinafter, the present invention will be described in detail by Examples. However, Examples below are only for the purpose of presenting the present invention, and the present invention is not limited to the Examples below.

EXAMPLE 1

[0068] Preparation of Mussel Adhesive Protein-Based Nanoparticles

[0069] 1-1. Preparation of Mussel Adhesive Protein fp-1

[0070] First, a variant of a mussel adhesive protein fp-1 (Mytilus mussel foot protein type 1) to which decapeptides (AKPSYPPTYK) had been repeatedly connected 12 times was prepared according to a publicly known procedure (See: Proc. Natl. Acad. Sci. U S A 2010, 107, 12850-3). The mussel adhesive protein fp-1 prepared as described above was allowed to be successfully expressed in E. coil, and then, it was produced through the purification and separation process using acetic acid.

[0071] 1-2. DOPA Modification Reaction

[0072] In order to obtain a mussel adhesive protein into which DOPA was introduced, a modification reaction using a tyrosinase enzyme (mushroom tyrosinase) was performed in vitro to convert tyrosine residues into DOPA. Specifically, 150 mg of a mussel adhesive protein and 5 mg of tyrosinase were added to 100 mL of a buffer solution consisting of 100 mM sodium phosphate, 20 mM boric acid and 25 mM ascorbic acid, and having a pH value of 6.8, and reacted for 1 hour. Thereafter, dialysis was performed using 5 L of a 5% acetic acid solution, followed by lyophilization to prepare a mussel adhesive protein into which DOPA was introduced.

[0073] 1-3. Preparation of Nanoparticles Using Mussel Adhesive Protein

[0074] Nanoparticles were prepared through electrospraying technology using the DOPA-introduced mussel adhesive protein fp-1 which had been obtained through Example 1-2. Specifically, the nanoparticles were dissolved in a solvent containing 2 wt % of MAP distilled water and ethanol at a ratio of 30:70, and then an FeCl.sub.3 solution or a VCl.sub.3 solution was added, followed by mixing so that the ratio of DOPA-Fe or DOPA-V became a molar ratio of 3:1. Thereafter, electrospraying was performed in a high voltage environment of 6 to 14 kV while injecting the solution at a rate of 1 mL/h using a syringe pump. The produced nanoparticles were collected in a phosphate-buffered saline (PBS, pH 7.4). The collected nanoparticles were analyzed using a scanning electron microscope (SEM), and the analysis results are shown in FIG. 2.

[0075] The specific preparation of the mussel adhesive protein is the same as that shown in Patent Application No. 10-2015-0035270, and the patent document is included in the present invention by reference as a whole.

[0076] 1-4. Analysis of Adhesive Properties of Nanoparticles Using Mussel Adhesive Protein

[0077] Adhesive properties of nanoparticles (FeMAP NPs) containing the prepared DOPA-Fe complex and nanoparticles (VMAP NPs) containing a DOPA-V complex were analyzed using a quartz crystal monitor sensor (QCM). After stabilizing the mussel adhesive protein for 10 minutes by administering distilled water to a mussel adhesive protein at a rate of 0.2 ml/min using a flow meter, a change in frequency was measured while administering 1 mg/ml of a nanoparticle solution to the mussel adhesive protein at the same rate for 10 minutes. Thereafter, the frequency change was measured while administering distilled water for washing to the mussel adhesive protein. In this case, bovine serum albumin (BSA)-based nanoparticles (BSA NPs) were used as a control group. As a result, the frequency changes of FeMAP NPs and VMAP NPs were about −115.82 Hz and −108.00 Hz respectively, showing a greater frequency change than −23.24 Hz, which was the frequency change of BSA NPs (FIG. 3). In addition, the frequency of BSA NPs was increased to −4.73 Hz during the washing process, whereas frequencies of FeMAP NPs and VMAP NPs were −114.44 Hz and −106.45 Hz respectively, indicating that there was no change in frequency, thereby confirming that the FeMAP NPs and VMAP NPs were continuously adhered to the surface.

EXAMPLE 2

[0078] Analysis of Photothermal Effects of Mussel Adhesive Protein-Based Nanoparticles

[0079] 2-1. Analysis of Absorbance of Nanoparticles

[0080] Absorbances in the near-infrared region of the FeMAP NPs and VMAP NPs prepared in Example 1-3 were analyzed through a UV-vis spectrometer, and the analysis results are shown in FIG. 4.

[0081] As shown in FIG. 4, when absorption spectrums were measured at 500 nm to 900 nm, a protein solution containing no DW, FeCl.sub.3, VCl.sub.3, and DOPA-metal complex showed almost no absorbance, whereas FeMAP NPs and VMAP NPs containing the DOPA-metal complex showed an increase in absorbance, and in particular, it was confirmed that the absorbances at 808 nm were 0.2838 and 0.4523 respectively.

[0082] 2-2. Analysis of Photothermal Effects of Nanoparticles

[0083] After 1 mL of FeMAP NPs and VMAP NPs prepared in Example 1-3 were each put into a cuvette, near-infrared rays were applied in a determined time period using an 808 nm laser having a power of 2 W/cm.sup.2. The temperature of the solution was measured using a thermometer at each time period and shown in FIG. 5

[0084] As shown in FIG. 5, it was confirmed that each of the nanoparticles generated heat when the near-infrared rays were applied and the temperature increased to 50° C. or higher within 10 minutes. Compared to FeMAP NPs, VMAP NPs exhibited also higher photothermal effects because absorbance in the near-infrared region was higher, and it was confirmed that the temperature increased to 50° C. or higher within 5 minutes. Accordingly, it was confirmed that the nanoparticles containing the DOPA-metal complex may be used as a photothermal agent in the near-infrared region.

EXAMPLE 3

[0085] Analysis of Gas Formation of Mussel Adhesive Protein-Based Nanoparticles

[0086] 3-1. Preparation of Photothermal-Responsive Nanoparticles Loaded with GSNO

[0087] Photothermal-responsive nanoparticles (VMAP @GSNO NPs) loaded with GSNO were prepared in the same manner as in Example 1-3. Specifically, the nanoparticles were dissolved in a solvent containing 2 wt % of MAP distilled water and ethanol at a ratio of 30:70, and then a VCl.sub.3 solution was added, followed by mixing so that the ratio of DOPA-V became a molar ratio of 3:1. Thereafter, 100 mM GSNO solution was added to the solution to be 40 μM, and then electrospraying was performed in a high voltage environment of 6 to 14 kV while injecting the GSNO solution-added solution at a rate of 1 mL/h using a syringe pump. The produced nanoparticles were put in a dialysis membrane of MWCO (molecular weight cut off) 3500, and then dialysis was performed using PBS (pH 7.4) to remove unloaded GSNO. Thereafter, the nanoparticles were analyzed using a scanning electron microscope (SEM), and the analysis results are shown in FIG. 6.

[0088] 3-2. Analysis of Photothermal-Responsive Nitric Oxide Gas Formation of Nanoparticles Loaded with GSNO

[0089] The photothermal-responsive nitric oxide gas formation of VMAP@GSNO NPs prepared in Example 3-1 was confirmed using a Griess reagent. 1 mL of an aqueous solution containing 9 mg/mL of VMAP@GSNO NPs was tubed into an MWCO 3.5 kDa membrane and cultured in 1 mL of PBS (pH 7.4). While applying a near-infrared laser at 1 hour intervals for 10 minutes, each solution was sampled and replaced with a new PBS solution. The formed nitric oxide gas was measured by mixing the sampled solution and the Griess reagent at a ratio of 1:1, and measuring the absorbance at 540 nm after 15 minutes. As a control group, a VMAP@GSNO NPs solution was sampled without applying a near-infrared laser. As a result, when the laser was not applied, the release of nitric oxide hardly occurred, but it was found that the release of nitric oxide occurred in the solution to which the laser was applied (FIG. 7).

EXAMPLE 4

[0090] Analysis of Drug Release Patterns of Mussel Adhesive Protein-Based Nanoparticles 4-1. Preparation of Photothermal-Responsive Nanoparticles Loaded with GSNO and Anti-Cancer Drugs at the Same Time

[0091] Photothermal-responsive nanoparticles (VMAP@GSNO/DOX NPs) loaded with GSNO and the anti-cancer drug doxorubicin (DOX) at the same time were prepared in the same manner as in Example 1-3. Specifically, the VMAP@GSNO/DOX NPs were dissolved in a solvent containing 2 wt % of MAP distilled water and ethanol at a ratio of 30:70, and then a VCl.sub.3 solution was added, followed by mixing so that the ratio of DOPA-V became a molar ratio of 3:1. Thereafter, the GSNO solution and the DOX solution were added to the solution, and then electrospraying was performed in a high voltage environment of 6 to 14 kV while injecting at a rate of 1 mL/h using a syringe pump. The produced nanoparticles were put in a dialysis membrane of MWCO (molecular weight cut off) 3500, and then dialyzed using PBS (pH 7.4) to remove unloaded GSNO and DOX. Thereafter, the nanoparticles were analyzed using a scanning electron microscope (SEM), and the analysis results are shown in FIG. 8.

[0092] 4-2. Analysis of Photothermal-Responsive Drug Release Patterns of Nanoparticles Loaded with GSNO

[0093] The photothermal-responsive drug release patterns of VMAP@GSNO/DOX NPs prepared in Example 4-1 were measured in vitro. 1 mL of an aqueous solution containing 9 mg/mL of VMAP@GSNO/DOX NPs was tubed into an MWCO 3.5 kDa membrane and cultured in 1 mL of PBS (pH 7.4). While applying a near-infrared laser at 1 hour intervals for 10 minutes, each solution was sampled and replaced with a new PBS solution. The amount of released DOX was measured through a fluorescence spectrum at an excitation wavelength of 485 nm and an emission wavelength of 580/10 nm. As a control group, a VMAP@GSNO/DOX NPs solution was sampled without applying a near-infrared laser. As a result, when the laser was not applied, the release of DOX hardly occurred, but it was found that the release of DOX occurred in the solution to which the laser was applied (FIG. 9).

EXAMPLE 5

[0094] Confirmation of Cytotoxicity and Anti-Cancer Effects of Photothermal-Responsive Nanoparticles

[0095] 5-1. Confirmation of Cytotoxicity of Photothermal-Responsive Nanoparticles

[0096] The cytotoxicity of the VMAP NPs and VMAP@GSNO NPs prepared in Examples 1-3 and 3-1 to human-derived breast cancer cells MCF7 (ATCC HTB-22) was confirmed. First, MCF7 cells were seeded in an amount of 1×10.sup.4 cells per well using a 48-well culture plate, and cultured at 37° C. in a humid atmosphere of 5% CO.sub.2 and 95% air for 1 day. Then, 9 mg/ml of each of the NPs was treated in a medium and cultured for 24 hours, and then cell viability was measured. Cell viability was determined by treating the CCK-8 reagent and performing a culturing process for 3 hours, and then measuring the absorbance at 450 nm from an aliquot of each medium (FIG. 10). Cells which had not been treated with nanoparticles were used as a control group.

[0097] As shown in FIG. 10, cell viabilities of VMAP NPs and VMAP@GSNO NPs were about 90 to 105% and 85 to 102% respectively, and it was confirmed that no cytotoxicity appeared compared to the control group.

[0098] 5-2. Confirmation of Anti-Cancer Effects of Photothermal-Responsive Nanoparticles

[0099] The cell viability of each cell according to the photothermal time of the photothermal-responsive nanoparticles was confirmed. Specifically, MCF7 cells were seeded in an amount of 1×10.sup.4 cells per well in a 48-well culture plate, and cultured at 37° C. in a humid atmosphere of 5% CO.sub.2 and 95% air for 1 day. Thereafter, 9 mg/ml of VMAP NPs, VMAP@GSNO NPs, and VMAP@GSNO/DOX NPs were treated in the medium and cultured for 30 minutes, followed by application of a near-infrared laser at 808 nm for 2 minutes, 5 minutes, and 10 minutes. Thereafter, a new medium was added and cultured for 24 hours, and then cell viability was measured using a CCK-8 reagent. Cells to which the laser was applied each hour without treatment with nanoparticles were used as a control group, and the results are shown in FIG. 11.

[0100] As shown in FIG. 11, as the time to apply the laser increases, the cell viability decreases, and when applied for 10 minutes, the anti-cancer effect according to the photothermal effect was confirmed by confirming that cell viabilities of VMAP NPs and VMAP@GSNO NPs were about 20% and 8.5% respectively. When the laser was applied for 10 minutes, the cell viability of VMAP@GSNO/DOX NPs was about 0.5%, and it was confirmed that trimodality therapy showed better anti-cancer effects through photothermal effect, nitric oxide gas, and anti-cancer drug.